Project Summary Herpes simplex virus type 2 (HSV-2) and bacterial vaginosis (BV) are two of the most prevalent infections of the female genital tract. Both infections are associated increased risk for HIV acquisition. While HSV-2 and BV have been epidemiologically linked in many studies, the direction and mechanism of their interaction is not known. Our group has pioneered daily participant-collected sampling to show that HSV-2 is detected (shed) frequently, on 10-28% of days, from genital surfaces, and that HSV-2 shedding is asymptomatic the majority of the time. More recent data has demonstrated that HSV-2 genital lesions and asymptomatic shedding episodes are associated with a persistent inflammatory cellular response, with IFN-? producing HSV-specific CD4+ and tissue resident CD8+ T-cells. These data suggest that HSV-2 infection chronically and fundamentally alters inflammation the genital tract. Drs. Marrazzo and Fredricks have similarly used frequent vaginal sampling and molecular methods to document rapid shifts in the vaginal microbiome, and to discover novel BV-associated bacteria (BVAB). This proposal seeks to elucidate the temporal and mechanistic nature of the HSV-2/BV association and to identify the components of the inflammatory response to HSV-2 that may increase the risk of BV. We hypothesize that frequent HSV-2 genital shedding causes increased levels of inflammatory cytokines, such as IL-1 and IFN-?, and decreased levels of antiviral innate immune molecules (i.e. secretory leukocyte protease inhibitor, SLPI) and that these changes result in fluctuations in the vaginal microbiome which predispose women to develop BV. To address these hypotheses, we will enroll 40 HSV-2 seropositive, HIV seronegative women with a history of BV in the past year who will self-collect genital and vaginal specimens daily for 30 days. We will also enroll 20 HSV-2 seropositive women without a clinical history of BV. We will quantify HSV using a validated PCR assay, and we will measure changes in the microbiome using gram stain (Nugent score), quantitative PCR (qPCR) and broad range 16S rRNA gene PCR with high throughput sequencing during HSV shedding episodes. Daily vaginal levels of innate immune proteins and cytokines associated with HSV-2 shedding will be measured. Relationships between HSV-2 shedding and Nugent score, quantity and presence of BVAB, and cytokine levels will be determined. These women will then be placed on suppressive acyclovir, which decreases HSV-2 shedding by 70-80%. We will determine whether suppression of HSV-2 shedding results in improved vaginal flora as measured by a decrease in Nugent score and increased concentration of lactobacilli. This integrated proposal will determine the relationship between HSV-2 shedding and the vaginal microbiome, and will give insight into the mechanistic basis of the association to provide a framework for appreciating how these infections influence inflammation within the genital tract. Ultimately, this proposal will contribute vital and novel information to the design of interventions that can modulate BV, and will provide us with knowledge to better promote women's reproductive health.